| The ideal result of periodontal therapy is not only to stop pathological progression themselves, but also to promote periodontal regeneration, thereby to achieve the complete recovery of the periodontal tissue physiologically and functionally. Bone Morphogenetic Proteins is kindred of cell cytokines which could highly induce osteogenesis. Among all the hypotypes, BMP-2 is a kind of strong bone-inducing factor and plays a crucial role in periodontal regeneration. But relying on the exogenous implantation only, the effective time and local concentration of BMPs are too limited to repair the bone injury, and to fulfill the goal of periodontal regeneration. Now, gene therapy technique has been introduced into the field of periodontology as a new hope to reach the target.The viral and non-viral delivers all have merits and demerits, and developing a novel and effective method to deliver gene becomes a new aim of the gene therapy research. Recently, some studies demonstrate that ultrasound-mediated microbubble destruction can enhance the transfection efficiency and expression of the exogenous gene to a certain orientation safely and effectively. This method would become a new breakthrough of the gene therapy.Therefore, this study planned to construct recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP containing the enhanced green fluorescence protein(EGFP) and the recombinant human bone morphogenetic protein-2 (rhBMP-2) by PCR, T/A cloning, investigate the transfection efficiency of rhBMP-2 gene in targeted cells by ultrasound-mediated microbubble destruction, and evaluate its feasibility of this method used in periodontal regeneration.Part 1: Construction of a Eukaryotic Expression Vector Containing the Enhanced Green Fluorescence Protein and the Recombinant Human Bone Morphogenetic Protein-2 Methods:1. A pair of primers specific for amplifying the DNA fragment encoding rhBMP-2 were designed and synthesized. The targeted DNA fragment was amplified from pIRES-rhBMP2 by PCR.2. rhBMP-2 and EGFP were inserted to the proper sites of vector pIRES, and internal ribozyme entry site (IRES) sequence was between the genes coding for rhBMP-2 and EGFP.3. The recombinant plasmid pIRES-rhBMP2-EGFP was first propagated in E.coli DH5α, and then was confirmed to contain hBMP2cDNA sequence by agarose gel electrophoresis and DNA sequence analysis.Results:The construction of the recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP and the correction of the open reading frame were confirmed through restriction enzyme maping analysis and DNA sequencing.Conclusion:By PCR, T/A cloning, the cDNA fragment encoding rhBMP2 and EGFP fragment can be cloned into pIRES to construct the recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP.Part 2: Ultrasound-Mediated Microbubble Destruction Enhances Exogenous Gene Expression in NIH3T3 Cells in VitroMethods:1. NIH3T3 cells were anabiosised and gone down to the 3rd to 4th generation, then cultured into 6 well plates.2. The cells were divided into 2 groups: plasmid DNA+ LipofectamineTM 2000 group(D+LF); plasmid DNA+ultrasound+microbubble group(D+U+M), and then plasmid DNA was transfected into cells with liposome or ultrasound and microbubble.3. 24～48 hours later, EGFP was applied to observe the expression of plasmid by fluoresence microscope, and the concentrations of BMP-2 were evaluated by enzyme-linked immunosorbent assay (ELISA).4. The results were analyzed by cure fitting and t-test of SPSS11.5. Results:The transfection efficiency rate is 7.30±1.58% in D+LF group, but 11.77±3.16% in D+U+M group(P<0.05); The concentration of BMP-2 in cells after transfection is 1164.35±724.67pg/ml in D+LF group, but 2932.70±656.27pg/ml in D+U+M group(P<0.05).Conclusion:Ultrasound-mediated microbubble destruction could enhance the transfection efficiency and expression of rhBMP-2 gene in NIH3T3 cells. This provides a new safe and effective gene delivery system for gene therapy in periodontal regeneration. |
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